Drugs which can block .beta.-adrenergic receptors are among the most valuable agents used presently in clinical medicine. In the United States the major drug employed clinically is propranolol, while numerous other agents are available commercially in Europe and probably will be used in the United States within the next few years. These drugs are used extensively throughout the world for the treatment of hypertension and angina pectoris. Dosage requirements can vary considerably among different patients. In part the variable dose requirement is related to differences in absorption and metabolism of the drug among individuals. Attaining the optimal dose is important in securing maximal therapeutic benefit and in avoiding potentially serious side effects of these drugs such as abnormalities in cardiac rhythm and blood pressure. It is generally felt that a simple and sensitive technique to measure these drugs in blood and other body tissues would facilitate the selection of optimal doses.
Detecting .beta.-adrenergic blocking drug levels in body fluids ideally should employ a technique which can be used with all of the agents. Moreover, it has been established with propranolol that a metabolite of the drug 4-hydroxypropranolol has therapeutic activity so that an ideal method should be able to measure pharmacologically active but not inactive metabolites in addition to the parent drug.
Presently available techniques include gas-liquid chromatography, fluorimetric procedures as well as radio-immunoassays. None of these have attained routine clinical use because of various technical problems. Most of these techniques are applicable for individual drugs rather than for the whole class of .beta.-blocking agents. Also such prior art methods do not specifically detect active metabolites.
.beta.-adrenergic blocking drugs were developed on the basis of their ability to antagonize the effects of adrenergic stimulating substances such as the natural neurotransmitter, norepinephrine or its analogue, isoproterenol, which has a uniquely high potency in stimulating .beta.-adrenergic receptors. In recent years it has been possible to label .beta.-adrenergic receptors in a variety of tissues using radioactive .beta.-blocking drugs (binder) such as .sup.3 H-dihydroalprenolol and .sup.125 I-hydroxybenzylpindolol. See the publications Lefkowitz et al, Biochem. Biophys, Res. Commun. 60:703-709, 1974; Aurbach et al, Science, 186:1223-1224, 1974. Neither of these publications nor any of several publications appearing in the succeeding years describing the binding of these and other radioactive drugs to the .beta.-receptor have disclosed anything beyond the fact that .beta.-adrenergic receptors can be measured with various radioactive forms of .beta.-blocking drugs, that .beta.-blocking drugs compete with the binding of these radioactive agents for the receptor, and that .beta.-adrenergic cardiac blockage is believed to be more closely correlated with free drug levels in the blood rather than total plasma blood levels. See McDevitt et al, Clin. Pharmacol. There, 20:152-157, 1976.
Moreover, the information contained in these above-mentioned publications does not provide a tool for measuring amounts of .beta.-blocking drugs in body fluids of human patients, because a number of needed elements, all of which were yet to be discovered, had to be discovered to exist for a successful assay for levels of .beta.-blocking drugs. For a successful assay for .beta.-blocking drug levels it was necessary to discover the nonspecific effects of body fluids on the binding properties of the .beta.-receptors and discover means of reducing or abolishing them. It was also necessary to discover that .beta.-blocking drugs added to body fluids could be recovered in a form that would still interact with the .beta.-adrenergic receptors. It was also necessary to show that in the presence of body fluids increasing amounts of .beta.-blocking drugs would in a predictable fashion produce progressively greater blockage of .beta.-receptors. Only after making a series of discoveries as disclosed herein which reduced nonspecific effects of body fluids on the .beta.-receptors, permitted recovery of added .beta.-blocking drugs and resulted in reproducible augmentations in receptor blockage with increasing amounts of .beta.-blocking drugs in body fluids was it possible to measure .beta.-blocking drugs and active metabolites in body fluids with this invention.